Method of determining blood-relationship by typing str alleles on the x chromosome and dna typing kit using the same

ABSTRACT

The present invention relates to a method for determining blood-relationship by typing STR alleles on the X chromosome and a DNA typing kit using the same, particularly when the father&#39;s DNA is not present. According to the method of the present invention, the blood-relationship of a suspected-grandmother and a suspected-granddaughter can be confirmed in the absence of the father&#39;s DNA by comparing STR alleles on the X chromosome of each subject. As for step-sisters born of different mothers, their blood-relationship can be determined by investigating whether they share STR alleles on the X chromosome when their mothers&#39; alleles on the X chromosome were excluded. Therefore, the method of the present invention can be usefully used in determining the blood-relationship in the absence of the father&#39;s DNA.

FIELD OF THE INVENTION

The present invention relates to a method for determiningblood-relationship by typing short tandem repeat (STR) DNA on the Xchromosome and a DNA typing kit using the same. More precisely, thepresent invention relates to a method for confirming blood-relationshipby comparing STR DNA on the X chromosome particularly when the father'sDNA is not present and a DNA typing kit using thereof.

BACKGROUND ART OF THE INVENTION

It has been known that only about 4% of human DNA is encoding functionalgenetic information and the rest of them have no such function. In caseof DNA without genetic information, a certain base sequence isrepeatedly shown or the same base sequence is given here and there onthe genome. When the certain base sequence is repeated within a region,it is called as “tandem repeat”. The non-informative DNA does not affecton cell function even when base sequence is changed by errors during thereplication process or by recombination since the DNA basically does nothave any genetic information. Thus, it can be said that the selectionpressure of the DNA is “zero” and accordingly lots of accumulatedchanges in base sequences can be found in them. Especially in case oftandem repeated base sequences, the repeating numbers of basic basesequence are vary, which is proven by the comparison test of the sameregion of DNA extracted from various people.

The variation of tandem repeated base sequence was first found byProfessor White at University of Utah (USA) in 1980 and laterforensically applied by Professor Jeffries at University of Leister(England) in 1985. Especially, it has been applied from then on toidentify a person using DNA, resulting in the birth of the term “DNAfingerprint”. The variation of repeating times is not serious and evenlimited in a tandem repeat genetic locus. However, when the variationshappening in tandem repeat genetic loci of various chromosomes areconsidered together, lots of peculiar combinations can be made. Forexample, a coin has only two sides: front and back. When it is thrown 10times, the chance of getting n times front or the chance of getting mtimes back is very few. But once chances are combined and permutation ismade, for example the chance of front in first throw and back in secondthrow, etc, the peculiarity is much increased. The certain combinationof tandem repeated alleles is called “DNA profile” and the DNA profileconsisting of 10 or more combinations of tandem repeated genetic loci isunique enough to identify each person on earth.

The variation of tandem repeat is huge but the variation gap betweengenerations is small as long as mutation is the cause. For example,there is not much difference of repeating times of tandem repeated basesequence between DNAs of father and son. Owing to this characteristic,tandem repeated DNA could be effectively used for the determination ofblood-relationship. It is now generalized throughout 80 or morecountries to decide whether an alleged father is real biological fatherby comparing DNA profiles and to identify a suspect by comparing his DNAwith the sample DNA taken from crime scene.

Tandem repeated allele was analyzed by Southern blot technology at theearly days. Precisely, genomic DNA was digested with a certainrestriction enzyme such as HaeIII and made into fragments, followed byelectrophoresis. Then, probe having base sequence that is hybridizedwith tandem repeated base sequence and marked with isotopes was used forthe reaction with the above DNA fragments, resulting in the presence oftandem repeated DNA fragments on the film. Fragments having same sizewere regarded as same alleles. In order to perform this method,comparatively large amount of specimen is required in addition to thetiresome process. Thus, this method is now limited in use and polymerasechain reaction (PCR) is replacing it. In the late 1980s, many STR basesequences that can be easily and precisely amplified by PCR were found,and therefore, more precise allele typing became possible. Furtherstudies and tests have been undergoing in the United States and FBI hadselected a standard test with 13 STRs and officially announced it as astandard for a genetic identification.

Exact typing of STR is very difficult without denaturing acrylamide gelelectrophoresis that has been used to decide base sequence since STR isgenerally no more than hundreds of base pair (bp) long. Denaturingacrylamide gel electrophoresis is very troublesome test method thatrequires complicated treatment procedures like silver staining ormarking with radioisotopes to show the fragments. In order to make thisdifficult experiment easy and simple, automatic gel electrophoresissystem or automatic sequencer was developed, with which fluorescencemarking was enabled and the test results were shown rightly. AppliedBiosystems, an American company, developed an automatic electrophoresissystem such as ABI310 possibly using 4 different fluorescent dyes, andPromega brought out a primer kit enabling up to 15 STRs to be typedsimultaneously by electrophoresis after marking each bundle of STRs tiedby 3-4 different sized STRs with different fluorescent dyes.

Though STR is very useful for identification or paternity test, it isnot very helpful when the alleged relationship is far from father andson or the amount of sample is small. For example, it is difficult todetermine the cousin-relationship by STR and to identify someone whenonly a hair fragment without its root is given.

Although mitochondria DNA typing is limited in use for the cases of amatrilineal heredity, it has been rapidly developed together with STR toidentify a biological mother or to analyze the infinitesimal quantity ofsample. A cell has hundreds to tens of thousands of mitochondria andeach mitochondrion has tens of thousands or hundreds of thousands ofDNA. Therefore, the mitochondria DNA outnumber nuclear DNA, so thatmitochondria DNA could be an easy target for genetic analysis. About 17kb-long mitochondria DNA is mostly encoding proteins or tRNA and hascomparatively small variation. However, a certain locus that is 1kb-long and is coding various regulation signals shows a big differenceamong people, from which it can be possibly confirmed whether thesubjects have same maternal line or even further identified whether thesubjects are derived from the same person.

While the maternal line can be confirmed by analyzing mitochondria DNA,the paternal line can be determined by typing STR on Y chromosome alongwith other STR. Except a short end, Y chromosome is transmitted to thenext generation without recombination, meaning that every maledescendants share the same Y chromosome except both ends if they havesame ancestor. Among many disclosed STRs from Y chromosome, about 10STRs that have comparatively more alleles and can be easily typed havebeen selected and used. STRs of Y chromosome have been importantly usedin finding male traces from a raped body and also can be an importantclue to decide whether descendents share the same ancestor even afterseveral generations.

Owing to their unique characteristics as shown above, mitochondria DNAand STR of Y chromosome became important tools for the identification ordetermination of blood-relationship even for the cases that cannot beconfirmed by STR of autosome and are also very useful for supplementingSTR of autosome. With all these methods, it is still difficult todetermine a distant relation over farther and son and hardly can get aresult from a test of real child without farther. Just in case thatbrothers or parents of the deceased father are alive, the relationshipcould be only indirectly presumed on the assumption that the deceasedfather surely had blood ties with them. If so, accurate results cannotbe guaranteed in most cases.

Thus, in order to solve the above problems, the present inventors havestudied and established the way to confirm the relationship of fatherand daughter by comparing STR of X chromosome in the absence of father'sDNA. And the present invention has accomplished by finding certain STRsof X chromosome that are useful for the identification and proving thatthe application thereof is successful.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method to confirmblood-relationship between a suspected-grandmother and asuspected-granddaughter by comparing STR alleles on the X chromosome ofeach subject in the absence of the father's DNA and/or to determineblood-relationship between sisters by investigating whether they shareSTR alleles on the X chromosome when their mothers' alleles on the Xchromosome were excluded.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a pedigree showing a typical family line required for theblood-relationship test by comparing STR alleles on the X chromosomes ofgrandmother and granddaughter,

FIG. 1 b is a pedigree showing a typical family line required for theblood-relationship test by comparing STR alleles on the X chromosomes ofsisters,

FIG. 3 is a diagram showing how X chromosome is transmitted fromgrandparents to grandchildren and how it is used for the determinationof blood-relationship,

A: The case of real children,

B: The case of non-real children

FIG. 3 is a diagram showing the location of selected 18 STRs on Xchromosome,

FIG. 4 is a diagram showing the steps of 18 STR alleles on X chromosome.

DETAILED DESCRIPTION OF THE INVENTION

To achieve the above objects, the present invention provides 18 STRalleles on X chromosome and a method to apply thereof for thedetermination of blood-relationship between a suspected-grandmother anda suspected-granddaughter or between suspected-sisters.

The present invention also provides a DNA typing kit, with which theabove STR alleles on X chromosome are effectively used.

Hereinafter, the present invention is described in detail.

The present invention provides 18 STR alleles on X chromosome and amethod to apply thereof for the determination of blood-relationshipbetween a suspected-grandmother and a suspected-granddaughter or betweensuspected-sisters.

Particularly, the present invention provides a method for investigatingSTR alleles on X chromosome of a women to see if there is a STR on Xchromosome transmitted from father when their mother's alleles on Xchromosome are excluded based on the fact that a man has only one Xchromosome and a father's X chromosome is transmitted to a daughter asit is, and for determining blood-relationship by comparing STR alleleson X chromosome of subjects in the absence of father's DNA.

The method for determining blood-relationship comprises the followingsteps:

-   -   1) extracting DNA from specimens of examinee;    -   2) obtaining massive STR DNA through PCR performed with STR on X        chromosome using the DNA obtained in the above step 1 as a        template;    -   3) investigating the physical, chemical characteristics of STR        DNA obtained in the above step 2;    -   4) determining blood-relationship by comparing types of STR        obtained in the above step 3.

In the above step 1, one or more components can be selected as specimensfrom a group consisting of blood, hair, saliva, epidermis, sperm,samples extracted from vagina, separated cells, tissue samples,dandruffs, ashes, etc, and mixed.

For the above step 2, it is preferable to select STR that has high rateof heteroconjugation on X chromosome and lots of alleles for betteridentification and to use standard allele steps obtained from cloning orPCR with whole alleles as molecular weight markers. Preferably selectedSTRs are DXS7133, DXS9895, DXS9898, DXS6807, DXS6803, DXS8378, DXS7132,DXS6789, GATA31E08, DXS9900, GATA144D04, GATA165B12, GATA186D06,GATA164A09, DXS6806, ATA28C05, DXS6804 or DXS6797.

It is also preferable to use multiplex PCR that amplifies simultaneously3 to 10 different sized STRs as a whole for simultaneous PCR andanalysis of many STRs. At this time, it is required to arrange primersequences for the amplification of each STR not to disturb each other.Since the distinguishment of alleles by electrophoresis depends on thesize of fragments, the size of STR alleles for electrophoresis is alsorequired not to be overlapped. Considering the limited fragmentseparating capacity of electrophoresis, 3-4 different sized STRs aresupposed to be filed for a one panel and marked with radioisotope orfluorescence.

The way of marking is to attach fluorescent material or radioisotope tothe 5′ end of a primer or to the 3′ end by taking advantage, of thereaction of terminal deoxynucleotidyl transferase after PCR. The otherway to confirm fragments without marking is to perform silver stainingafter electrophoresis. It is possible to mark various multiplexingpanels with all different colored fluorescent materials owing to theirvarious wavelengths. It is also possible to trace fragments marked byvarious fluorescent materials using automatic electrophoresis systemsuch as ABI310 provided by Applied Biosystems.

Each STR alleles show different repeating times, resulting in thedifference in length of amplified fragments. Thus STR alleles can beidentified by measuring their length by electrophoresis. The way tomeasure the length by electrophoresis is to measure migration distanceof a fragment per time unit or to measure time that takes for a fragmentto move a certain distance when automatic electrophoresis system isused. The migration induced by electrophoresis is influenced not only bythe length of a fragment but also by the structure or sequence of thefragment, so that standard alleles should be used as a criterion forcomparison in order to determine correct alleles. Therefore, usingallele steps is essential for genetic identification by STR. The presentinvention provides the allele steps of 18 STRs.

The above step 3 is for the analysis of physical and chemicalcharacteristics of the extracted DNA. Precisely, this stage includes theprocedure of electrophoresis that measures the length of DNA fragmentsamplified by PCR, method to use mass spectrometer to measure the mass ofthe amplified fragments, method to measure the differences among basesequences caused by hybridization and method to determine the basesequence directly. In order to investigate hybridization, it is includedto use DNA array representing the characteristics of sample DNA byattaching the established standard DNA whose properties are alreadydisclosed like DNA chip to the surface of a matrix for further reactionwith the sample DNA.

In order to investigate physical and chemical characteristics of DNA inthe above step 3, a certain device that is able to perceive a markattached to sample DNA is necessary, otherwise DNA fragments should betreated to be detected well. Autoradiography and scintillation countingmethods are preferably used to detect DNA marked with radioisotopes, ABIautomatic sequencer provided by Applied Biosystems or FMBIO provided byHitachi is useful for detecting DNA marked with fluorescent materials,and silver staining method is suitable for detecting unmarked DNA.

The method for determining blood-relationship of the present inventionis based on the fact that a man has only one X chromosome that istransmitted to his daughter directly. Thus, when X-STR that is sharedwith her mother is excluded, the remaining X-STR of daughter is sharedwith her father, and the father's X-STR is transmitted from grandmother.Repeatedly speaking, one of the two X chromosomes of grandmother istransmitted to father and then it is transmitted to daughter as it is.Therefore, grandmother and granddaughter share half of X-STR each otherif they are in a lineal relation. FIG. 1 a is a diagram showing thetransmission mode of X chromosome. In this diagram, a rectanglerepresents a man and a circle represents a woman. An oblique line on arectangle means he is deceased. Each generation is represented by theRoman numbers I, II, III, etc, and each individual of each generation isrepresented by the Arabic numbers. Father(II-1)'s X chromosome is notexactly same as one of the two X chromosomes of grandmother(I-2) sinceSTR alleles of grandmother are exchanged each other by the recombinationof her two X chromosomes during the meiosis. However, half ofgranddaughter(III-1)'s STR alleles on X chromosome are exactly same asthose of grandmother in case they are in a direct line.

More precisely, as comparing grandmother(I-2)'s DNA with that ofsuspected-granddaughter(III-1) as seen in FIG. 2, the consistency of 8repetition on a specific STR suggests they are in a direct line(A), butif the number of repetition is not in accord, it can be determined thatthey are not in a direct line(B). The black bar in FIG. 2, II-1represents Y chromosome. If grandmother (I-2) s X chromosome istransmitted to granddaughter(III-1) by way of father(II-1), every STRalleles on father's X chromosome should be reflected in granddaughter'sX chromosome. If grandmother and granddaughter are not in a bloodrelationship, each STR allele has much chance to be different each otherand most STR hardly have chance to have consistent alleles continuously.Therefore, blood-relationship can be determined by whether STR allelesare shared by subjects; that is, when most of STR alleles of subjectsare repeatedly consistent, it can be determined that they are in ablood-relationship.

The way to confirm a blood-relationship by comparing STR on X chromosomecan be applied for determining a blood-relationship betweensuspected-sisters. FIG. 1 b is a pedigree of a family that has 3daughters. X-STR of a woman is made up of one transmitted from fatherand the other from mother. Thus, when X-STR alleles transmitted frommother are excluded, the rest alleles must be in accord with X-STRalleles of father. It is all the same to every sister, meaning theyshare the same X-STR alleles when alleles from mother are excluded ifthey have a same biological father.

In order to confirm a real child of a deceased father by the method ofthe present invention, it ought to be investigated whether grandmother'sX-STR alleles were transmitted to granddaughter through father. If thesubjects are not in a blood-relationship, the chances are very low forthem to have same alleles.

Therefore, the present inventors have confirmed whether the X-STRalleles of grandmother were transmitted to granddaughter.

At first, the inventors have randomly selected 18 STRs evenly spread onX chromosome, and analyzed the alleles of those 18 STRs. As a result, atleast one of granddaughter's alleles has been found in grandmother'salleles. And granddaughter has been confirmed to have the same allelesas grandmother's alleles when alleles transmitted from mother wereexcluded, proving that half of grandmother's X-STR alleles have to betransmitted to granddaughter through father (see Table 1). TABLE 1 STRprofile of X chromosomes of grandmother and granddaughter Size of PCRFragment Grand- Gene Profile STR mother Granddaughter GrandmotherGranddaughter DXS9898 201/205 201/205 6 7 6 7 DXS6807 252/264 252/264 25 2 5 DXS6803 114 114/118 2 2 2 3 DXS8378 210/214 206/214 3 4 2 4DXS7132 283/287 287 3 4 4 4 DXS6789 119/136 136 3 7 7 7 GATA31E08 243239/243 5 5 4 5 DXS9900 122/126 122/126 4 5 4 5 GATA144D04 237 237/249 33 3 6 GATA165B12 125/134 125/129 1 3 1 2 GATA186D06 205/209 200/208 3 42 4 GATA164A09 246/250 250 5 6 6 6 DXS6806 167/171 167 2 3 2 2 ATA28C05279/288 279/282 3 6 3 4 DXS6804 187 178/187 6 6 4 6 DXS6797 259 259/2632 2 2 3

Next, the present inventors have also confirmed that there is almost nochance for subjects to have the same alleles in case they are not in ablood-relationship.

Although alleles are many and various, there is still a chance to havethe same alleles on a specific STR even between the two people not in ablood-relationship. Each target STR for genetic identification generallylocates on a different chromosome. Thus, the STRs are transmitted to adescendent independently since they are not linked. Generally used STRsets are reported to have reached Hardy-Weinberg Equilibrium and LinkageEquilibrium since they locate on different chromosomes. Therefore, thepossibility that STR alleles are accidentally consistent betweensubjects is calculated by multiplying the frequency of each STR alleles.However, as for X-STR alleles, they locate on a same chromosome andsupposed to be in a linkage even though DNA could be changed betweenhomologous chromosomes by recombination. Thus, it is difficult toestimate the possibility of accidental accordance of X-STR alleles bymultiplying their frequencies.

The present inventors have obtained the DNA profiles representing thecombination of 18 X-STR alleles for 59 Korean women to investigate thepossibility that granddaughter's alleles from father can be accidentallyincluded in grandmother's DNA profile (see Table 2 and 3). TABLE 2 18 XSTR profiles of Korean women DXS7133 DXS9895 DXS9898 DXS6807 DXS6803DXS8378 DXS7132 DXS6780 GATA31E08 1 5 5 2 3 6 8 1 1 3 3 2 2 2 5 3 8 3 32 4 5 2 4 5 6 4 3 2 2 2 2

5 2 3 1 5 3 4 4 2 2 6 7 1 1 2 4 3 4 5 6 8 8 6 5 4 4 4 1 1 6 6 1 4 3 3 24 4 5 7 9 4 3 5 4 5 1 3 6 7 1 4 3 3 2 3 4 5 3 3 5 5 6 4 4 1 3 6 7 4 4 23 3 3 4 5 8 10 4 5 7 4 4 1 1 6 7 1 5 2 3 2 3 3 5 7 8 4 6 8 4 4 1 2 6 7 14 2 3 3 4 4 5 3 8 4 5 9 4 6 2 3 6 6 4 6 2 2 2 4 3 5 3 8 1 5 10 4 6 2 2 67 1 5 3 3 3 4 5 5 2 7 3 3 11 4 4 1 3 6 8 1 5 2 2 3 3 4 5 7 8 3 5 12 4 42 3 6 8 4 6 2 2 2 4 4 4 2 7 4 5 13 5 7 2 2 7 7 5 5 3 3 2 2 3 4 2 2 1 314 4 6 2 3 6 6 4 5 2 3 2 3 3 4 3 4 4 5 15 5 6 2 2 3 6 1 4 2 2 2 4 2 5 23 5 5 16 4 6 1 4 6 7 1 4 2 3 2 3 4 4 2 3 3 3 17 4 4 1 1 6 7 1 4 3 3 2 23 3 7 10 2 5 18 4

2 2 6 8 1 1 2 2 4 4 3 5 7 9 3 4 19 4 5 2 2 6 6 1 5 1 3 3 3 2 4 2 8 4 520 4 4 1 1 6 7 3 4 2 2 2 3 4 4 3 8 3 5 21 4 4 1 1 6 7 4 6 2 2 2 2 3

3 3 4 5 22 4 4 1 1 6 7 1 4 3 3 2 2 2 3 4 7 1 5 23 4 4 1 1 7 7 1 1 2 2 33 2 3 3 7 3 6 24 4 4 2 2 5 6 1 4 2 6 2 3 3 5 3 7 3 3 25 5 5 1 1 6 7 4 53 4 2 2 2

3 7 4

26 4 4 2 2 6 6 1 1 2 2 2 3 4 5 9 9 1 6 27 4 4 2 2

7 1 4 1 4 2 3 5 5 3 3 5

28 4 5 1 1 5 6 1 5 2 3 2 4 4 4 2 7 3

29 4 4 1 1 6 6 1 1 2 2 2 2 3 5 3 8 1 3 30 4 4 2 2 3 7 1 4 2 3 2 2 4 4 22 5 5 31 4 4 2 2 8 8 1

1 3 2 3

4 8 8 4 4 32 4 5 1 1 3 7 4 3 3 3 2 2 5 5 8 8 4 4 33 4 4 2 2 7 8 4 4 2 32 2 4 6 6 7 3 5 34 5 5 1 2 6 7 1

3 3 2 2 3

2 3 4 4 35 4 4 2 4 6 6 1

3 3 4 3 4 2 2 5 6 36 4

1 3 7 7 1 1 1 3 2 3 3 4 8 8 5 6 37 5 6 1 3 6 7 1 4 2 3 2 2

5 8 9 3 4 38 4

1 2 6 8 4 4 2 2 2

3 3 3 7 1 4 39 5 6 3 4 6 7 3 4 1 1 2

8 8 6 6 40 4

1 4 6 7 4 6 2 3 2 3 2 5 2 9

41 4

1 1

8 1 4 1 4 2

5 6 2 3 4

42 4 4 1

6 6 1 5 3 4 4 5 2 3 2 8 1 3 43 4

1

6 7 1 1 3 3 2 2 3 3 3 8 4 4 44

4 1 2 5 7 1 4 2 3 2 3 2 3 3 3 4 5 45 4 4 2 4 6 7 4 4 2 3 2 3 6 6 6 6 5 546 5

1 3 7 7 1 4 3 3 2 3 3

3 3 5 6 47 4 4 2 2 3 9 1

2 4 2 3 3 4 2 2 3 3 48 4

1 1 6 7 1

2 3 3 3 2 4 3 3 4 4 49 4 4 1 1 6 8 1 1 2 2 2 3 4 4 3 7 5 6 50 4 5 1 3 88 5

1 2 2 3 4 5 2 8 3 4 51 4 4 2 4

6 4 5 2 4 2 2 4 3

8 3

52 4 5 1 5 7 8 5 6 2 3 2 2 6 6 7 8

6 53 4 4 3 5 6 7 4

3 3 2 3 4

3 7 3

54 4

1 2 3 7 1 4 2 2 2 2 4 4 7 7 2 2 55 4

2 4 6 7 1 4 2 3 2 4 3 6 3 3

6 56 4 4 1 1 6 6 4 4 2 2 2 2 4 4 4 8 3

57 4 6 3 4 6 7 4 5 2 2 2 2 4 6 8 10 3 3 58 4

1 3 7 7 1 1 2 4 2 3

7

4 6 59 5

3 6 6 2 4 3 3 2 3 2 4 2 4 3

TABLE 3 18 X STR profiles of Korean women DXS9900 GATA144D04 GATA165B12GATA186D06 GATA164A09 DXS6806 ATA28C05 DXS6804 DXS6797 1 5 6 3 4 1 2 3 46 6 2 2 2 4 4 7 4 7 2 5 6 3 5 2 2 4 4 3 6 2 2 6 7 4 6 3 4 3 6 6 3 6 2 33 4 2 6 2 3 4 7 5 5 3 5 4 5 5 3 5 1 2 3 4 4 6 2 2 4 4 5 5 4 5 5 5 5 4 51 2 3 4 6 6 3 3 3 5 5 5 5 5 6 4 5 3 4 2 3 3 3 2 2 2 2 4 6 7 7 3 5 7 5 51 3 2 3 4 4 3 6 2 3 3 4 4 6 3 6 8 3 6 3 5 1 2 3 3 4 6 2 2 3 6 3 8 3 3 93 4 3 4 1 2 3 4 1 2 2 2 4 6 6 7 6 6 10 5 7 3 6 2 4 3 4 2 7 2 3 4 6 6 6 46 11 3 5 3 4 1 2 4 5 3 5 2 2 4 4 6 7 4 5 12 3 5 2 4 1 2 3 3 1 2 2 2 3 64 6 4 6 13 6 6 3 4 2 2 4 4 1 1 1 3 6 6 6 7 4 7 14 4 4 4 6 2 2 3 5 3 7 22 4 6 6 8 4 6 15 5 5 3 3 2 2 3 3 2 4 2 5 4 6

7 4 6 16 6 7 2 4 1 3 4 5 2 7 2 6 5 6 4 7 6 5 17 1 6 3 4 2 2 3 3 6 6 1 24 6 6 6 3 4 18 5 6 4 4 1 3 4 4 1 5 2 2 4 6 4 6 3 2 19 5 5 4 4 1 1 3 4 66 2 4 3 6 5 5 3 6 20 5 6 3 3 1 2 4 5 2 6 6 5 6 6 6 6 4 5 21 6 6 3 5 1 24 4 6 6 2 2 6 6 4 6 2 4 22 5 5 4 4 2 2 3 4 2 6 2 3 6 6 4 4 4 4 23 3 5

6 2 2 3 4 2 2 2 2 3 6 4 5 3 4 24 4 5 2 4 2 2 3 4 2 4 2 3 4 6 4 4 3

25 4 4

5 3 3 4 4 1 6 2 2 4 5 4

3 5 26 6 6 3 3 1 3 4 4 6 6 2 5 4 6 4 6 3 4 27 1

4 6 2 2 4 6 2 5 2 2 6 6 4 5 6 6 28 5 6 3 6 1 2 3 3 1 7 2 2 4

4 6 4 5 29 1

3 3 1 4 4 4 1 7 2 3 3 4 4 7 4 5 30 4 4 4 4 2 3 3 3 1 1 2 4 5 6 4 6 5 531 6 6 4 4 2 4 3 4 2 6 2 4 6 6 4 6 4 4 32 5 5 3 4 1 2 4 4 1 7 3 3 4 6 66 3 4 33 5 6 3 4 2 3 3 4 6 8 4

6 6 4 6 3 4 34 3 5 4 5 3 3 3 4 3 6 2 2 3 6 4 5 5 6 35 5 5 3 3 1 2 4 5 36 2 3 6 6 1 7 2 4 36 6 6 3 3 1 3 3 3 2 6 2 2 6 6 3 7 3 4 37 5 5 3 4 2 32 4 6 6 2 3 4 4 4 4 5 5 38 5 6 3 4 1 2 3 5 6 6 2 3 6 6 6 6 3 5 39 6 6 66 2 3 4 4 2 6 2 2 3 6 5 5 4 5 40 4 4 3 4 2 2 4 5 2 2 2 3 4 6 6 7

5 41 1 5 2 4 3 3 3 4 2 3 2 2 6 6 6 6 4 5 42 5 6 1 5 2 2 3 3 1 2 2 2 6 64 4 4 4 43 4 5 2 3 2 3 1 3 3 3 2 2 4 4 7 8 4 6 44 1 5 3 5 2 2 4 4 1 6 23 6 6 7 8 3 5 45 2 2 4 4 2 2 3 4 1 3 2 2 3 4 4 6 4 5 46 5

3 3 2 2 4 4 2 2 4 4 4 4 5 7 5 6 47 3 3 1 1 2 3 3 4 2 6 3 5 2 6 4 6 4 548 6 6 1 4 1 2 3 6 2 3 2 2 4 6 7 7 4 4 49 8 6 3 3 1 2 4 5 1 6 2 2 3 6 46 3 5 50 5 5 4

2 3 3 5 2 6 2 3 6 6 7 8 4 4 51 1 5 3 5 1 3 3 3 6 6 2 2 4 6 6 7 5 6 52 36 3 6 2 2 4 4 1 6 2 3 5 6 7 8 4 5 53

6

1 2 2 3 3 4

6 5 3 5 4 7 4 5 54 5

3 3 1 3 4 4 2 2 2 2 6 7 4 6 4 4 55 6 6 3 3 2 3 3 3 1 1 2 4 4 6 4 4 4 656 4 5 3 4 2 2 3 3 1 1 2 2 4 4 7 7 5 5 57 4 4 4 6 2 2 2 4 1 2 3 4 3 4 57 3 4 58 5 5 4 6 2 2 4 6 6 6 2 4 4 6 5 7 6 6 59 5 5 3 4 2 4 3 4 1 2 3 62 3 6 6 3 5

Presuming the No 1 of the Table 2 and 3 as grandmother(suspected-grandmother), the possibility of granddaughter-cannot-be wasanalyzed for all the rest people. Again, presuming the No 2 of the Table2 and 3 as grandmother, the possibility of granddaughter-can-be wasanalyzed for all the rest people. Precisely, if any STR that is notaccord with alleles of suspected-grandmother is included among 18 STRsof suspected-granddaughter, there is no chance of blood-relation betweenthem. Meanwhile, if every alleles of suspected-granddaughter are sharedon grandmother's alleles, there is high chance of blood-relation betweenthem. The present inventors performed the same test as above with DNAprofiles of 59 people. As a result, 1711 pairs could be analyzed andthere was just one case that suspected-granddaughter was proved to bereal granddaughter.

Therefore, the chance that suspected-granddaughter's STR alleles areaccidentally consistent with those of suspected-grandmother, so that sheis regarded as a biological daughter of deceased father is probably1/1711, which means that the method for determining blood-relationshipby typing STR alleles on X chromosome of the present invention istrustworthy.

The present invention also provides a DNA typing kit that could beeffectively used for determining blood-relationship betweensuspected-father and suspected-daughter.

DNA typing kit of the present invention includes a container of primersenabling to amplify X-STR. In addition, the kit also includes molecularweight marker that consists of standard alleles DNA of each X-STR.Factors that are used for providing proper conditions for PCRamplification such as buffer solution, amplification enzyme,nucleotides, etc, can be further included. The preferable STR for thepresent invention is the one that locates on X chromosome and isdistributed evenly. One or more STRs selected from a group consisting ofDXS7133, DXS9895, DXS9898, DXS6807, DXS6803, DXS8378, DXS7132, DXS6789,GATA31E08, DXS9900, GATA144D04, GATA165B12, GATA186D06, GATA164A09,DXS6806, ATA28C05, DXS6804 and DXS6797 could be used. Primers that areable to amplify STRs used for test selectively are used for the DNAtyping kit of the present invention and especially, primers shown inTable 4 can be preferably used. TABLE 4 Base sequences of primers andSTRs of X chromosome Locus Direction Primer Sequence 1 DXS7133 F SEQ.ID. No 1 R SEQ. ID. No 2 2 DXS9895 F SEQ. ID. No 3 R SEQ. ID. No 4 3DXS9898 F SEQ. ID. No 5 R SEQ. ID. No 6 4 DXS6807 F SEQ. ID. No 7 R SEQ.ID. No 8 5 DXS6803 F SEQ. ID. No 9 R SEQ. ID. No 10 6 DXS8378 F SEQ. ID.No 11 R SEQ. ID. No 12 7 DXS7132 F SEQ. ID. No 13 R SEQ. ID. No 14 8DXS6789 F SEQ. ID. No 15 R SEQ. ID. No 16 9 GATA31E08 F SEQ. ID. No 17 RSEQ. ID. No 18 10 DXS9900 F SEQ. ID. No 19 R SEQ. ID. No 20 11GATA144D04 F SEQ. ID. No 21 R SEQ. ID. No 22 12 GATA165B12 F SEQ. ID. No23 R SEQ. ID. No 24 13 GATA186D06 F SEQ. ID. No 25 R SEQ. ID. No 26 14GATA164A09 F SEQ. ID. No 27 R SEQ. ID. No 28 15 DXS6806 F SEQ. ID. No 29R SEQ. ID. No 30 16 ATA28C05 F SEQ. ID. No 31 R SEQ. ID. No 32 17DXS6804 F SEQ. ID. No 33 R SEQ. ID. No 34 18 DXS6797 F SEQ. ID. No 35 RSEQ. ID. No 36

Standard alleles DNA used as a molecular weight marker could be obtainedby amplifying genome DNA having each alleles through PCR, by cuttingcloned alleles with restriction enzymes or by synthesizing artificialnucleic acid.

DNA typing kit of the present invention can also include STR primerattached with signaling material selected from a group consisting ofdioxygenin, biotin, radioisotope, fluorescent material, enzyme, andantibody. The examples of the above enzyme are peroxidase, alkalinephosphatase, luciferase, etc, and FITC and TRITC can be used as afluorescent material. As a coupler, 4-chloro-1-naphtol (4CN),diaminobenzidine (DAB), aminoethyl carbazole (AEC), 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine(OPD) or tetramethyl benzidine (TMB) can be used.

The above X-STR alleles can be provided as the form of DNA sequencefixed on a supporting material. Precisely, the present inventors had 5′end or 3′ end of X-STR alleles DNA fixed using avidin-biotin conjugationor antigen-antibody binding, or had DNA chain fixed on the surfacecontaining amine group.

In order to prepare DNA typing membrane or chip, every STR alleles thatare the targets for typing, are amplified by PCR and denaturalized, fromwhich single stranded STR alleles are obtained and then fixed onto thesupporting material. The primer used for the amplification of STRalleles with PCR has specific material or functional group attached onthe 5′ end of a forward primer or a backward primer. At this time,biotin, primary amine, digoxigenin or fluorescent material can be usedas specific material or functional group. PCR was repeatedly performed25-40 times using a primer having specific material or functional groupattached thereon and a normal primer to amplify STR alleles up to10⁵-10⁷ fold. Such amplified STR alleles were heat-denaturalized toobtain single stranded STR alleles. At last, the single stranded STRalleles attached on DNA typing membrane or chip was prepared by reactingspecific material or functional group attached on single stranded STRalleles selected among every obtained single stranded STR alleles andsupporting material at 60° C. for 1 hour.

The supporting material of DNA typing membrane or chip on which STR isfixed includes nylon membrane, nitrocellulos membrane, glass slide,polycarbonate or synthetic resins, etc. And, avidin, streptavidin,aldehyde group, etc, can be spread on the surface of the supportingmaterial.

As for membrane is used as a supporting material, biotin of STR allelesis combined with avidin or streptoavidin attached on the membrane, sothat STR alleles are fixed on the membrane, resulting in the preparationof DNA typing membrane. As for glass slide is used as a supportingmaterial, biotin of STR alleles is combined with avidin or streptoavidinattached on glass slide or primary amine of STR alleles is combined withaldehyde group attached on glass slide, resulting in the preparation ofDNA typing chip.

EXAMPLES

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Method for Confirming Real Child by Comparing X-STRs ofGrandmother and Granddaughter in the Absence of the Father's DNA

In order to confirm a blood-relationship by comparing X-STRs ofgrandmother and granddaughter in the absence of father's DNA, thepresent inventors have selected following STRs from US GenBank; DXS7133,DXS9895, DXS9898, DXS6807, DXS6803, DXS8378, DXS7132, DXS6789,GATA31E08, DXS9900, GATA144D04, GATA165B12, GATA186D06, GATA164A09,DXS6806, ATA28C05, DXS6804 and DXS6797. The above STRs are located on Xchromosome, have high rate of heteroconjugation, and are distributedevenly on X chromosome.

Next, the present inventors have investigated the frequencies of allelesof 200 Korean women to estimate the kind and the frequency of the above18 STR alleles. 5′ end of only one sides of 18 primer pairs eachrepresented by SEQ. ID. No 1 through No 36 was marked with fluorescentmaterial (6-FAM). These primers were used for the amplification of STRalleles. The sizes and the numbers of amplified alleles were shown inTable 5. TABLE 5 Sizes of PCR-amplified fragments of STR alleles used inthe present invention No DXS7133 DXS9895 DXS9898 DXS6807 DXS6803 DXS8378DXS7132 DXS6789 GATA31E08 1 100 141 180 249 110 200 276 113 227 2 104145 184 253 114 204 280 117 231 3 108 149 188 257 118 208 284 121 235 4112 154 192 261 122 212 288 125 239 5 116 158 196 265 126 216 292 129243 6 120 200 269 130 296 133 247 7 124 204 102 300 137 8 208 106 141 9212 145 10  149 11  153 No DXS9900 GATA144D04 GATA165B12 GATA186D06GATA164A09 DXS6806 ATA28C05 DXS6804 DXS6797 1 108 230 125 192 229 163273 166 243 2 112 234 129 200 233 167 276 170 251 3 116 238 133 204 237171 279 174 259 4 120 242 137 208 241 175 282 178 263 5 124 246 141 212245 179 285 182 267 6 128 250 216 249 183 288 186 271 7 132 254 220 253291 190 275 8 257 294 194 279<1-1>DNA Extraction

In order to compare X-STR of grandmother with that of granddaughter, thepresent inventors have extracted DNA from blood of grandmother,granddaughter and granddaughter's mother. Particularly, 500 μl of bloodwas transmitted into a test tube containing EDTA, a kind ofanticoagulant. The test tube was centrifuged for 1 minute and thesupernatants were discarded. Added 900 μl of ACE solution (NH₄Cl 8 g,Na₂EDTA 1 g, KH₂PO₄ 0.1 g, pH 7.0/liter) thereto, vortexed thereof for15 seconds, and mixed thereof by stirrer at room temperature at 30 rpmfor 10 minutes. Centrifuged thereof for 1 minute, and the supernatantswere discarded. The pellets were resuspended in 300 μl of nuclei lysisbuffer (10 mM Tris-HCl, pH 8.0, 400 mM NaCl, 2 mM EDTA). Added 20 μl of10% SDS and 6 μl of protease K (20 μg/μl) thereto and mixed thereofwell. Cultured thereof at 56° C. for 2 hours. Added 100 μl of saturatedNaCl into the tube, vortexed thereof for 15 minutes, and left thereof atroom temperature for 5 minutes. Centrifuged for 2 minutes andtransferred the supernatants into a new test tube. Added two volumes ofalcohol into the tube. After closing the tube, shaken the tube up anddown slowly about ten times. Transferred floating nucleic acid clotformed in the above test tube into a new test tube containing 500 μl ofTE buffer (10 mM Tris-HCl, pH8.0, 1 mM EDTA). Finally, extracted DNA byleaving thereof at 56° C. water bath for overnight. Extracted DNA wasquantified with DynaQuant (Hoefer) using fluorescent assay.

<1-2>Multiplexing Amplification of X-STR

In order to amplify STR of DNA extracted in the above Example <1-1>,multiplexing PCR was performed using primer pairs represented by SEQ.ID. No 1-36. STRs were sorted 3 multiplex panels (the first panel:DXS7133, DXS9895, DXS9898, DXS6807, GATA165B12 and DXS6797; the secondpanel: DXS6803, DXS6806, DXS8378, GATA144D04, DXS7132, DXS6789,GATA186D06, GATA31E08 and ATA28C05; the third panel: GATA172D05, DXS6804and GATA164A09), and then PCR was performed.

Particularly, reaction buffer was made with 2 ng of DNA extracted in theabove Example <1-1>, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, dNTP 200 μM,0.2 μM of each primer (DXS8378 and ATA28C05: 0.3 μM), 1.5 mM of MgCl₂and 2.5 U of DNA polymerase, and final volume of the reaction buffer wasadjusted to 25 μl with distilled water. Amplification was performed by30 cycles as follows: a denaturing step at 94° C. for 1 minute, a primerannealing step at 56° C. for 1 minute and an extension step at 72° C. 1minute. In case of need, the PCR condition was modified throughadditional experiments.

<1-3> Physical and Chemical Characteristics of Amplified STR

DNA amplified in the above Example <1-2>was analyzed with ABI310, anautomatic electrophoresis device, and the results were shown in Table 6.TABLE 6 STR profiles of grandmother, granddaughter and granddaughter'smother Size of PCR Fragment Gene Profile STR Grandmother GranddaughterMother Grandmother Granddaughter Mother DXS 114 110/ 110/ 5 5 4 5 4 67133 114 118 DXS 140 140 140 1 1 1 1 1 1 9895 DXS 201/ 201/ 201 6 7 6 76 6 9898 205 205 DXS 252/ 252/ 252/ 2 5 2 5 2 5 6807 264 264 264 DXS 114114/ 114/ 2 2 2 3 2 3 6803 118 118 DXS 210/ 206/ 206/ 3 4 2 4 2 3 8378214 214 210 DXS 283/ 287 287 3 4 4 4 4 4 7132 287 DXS 119/ 136 136 3 7 77 7 7 6789 136 GATA3 243 239/ 239 5 5 4 5 4 4 1E08 243 DXS 122/ 122/105/ 4 5 4 5 1 4 9900 126 126 122 GATA1 237 237/ 245/ 3 3 3 6 5 6 44D04249 249 GATA1 125/ 125/ 125/ 1 3 1 2 1 2 65B12 134 129 129 GATA1 205/200/ 200/ 3 4 2 4 2 3 86D06 209 208 204 GATA1 246/ 250 230/ 5 6 6 6 1 664A09 250 250 DXS 167/ 167 167/ 2 3 2 2 2 3 6806 171 171 ATA 279/ 279/282/ 3 6 3 4 4 5 28C05 288 282 285 DXS 187 178/ 178 6 6 4 6 4 4 6804 187DXS 259 259/ 259/ 2 2 2 3 2 3 6797 263 263<1-4>Comparison of STR Types

Table 6 is showing 18 X-STR gene profiles of granddaughter, grandmotherand mother. When mother's genotypes are excluded from granddaughter's,the alleles indicated in dark letters in Table 6 are left. When the twoalleles are identical, both alleles are indicated in same dark letters.In order to determine blood-relationship between grandmother andgranddaughter, the alleles indicated in dark letters, in other wordsalleles of the paternal line, seen in granddaughter's gene profileshould be included in grandmother's gene profile. As seen in Table 6,alleles of the paternal line were all found in grandmother's geneprofile, resulting in the confirmation of the fact that granddaughterwas a real child of a deceased father.

Example 2 Method for Confirming Real Child by Comparing X-STR of Sistersin the Absence of the Father's DNA

<2-1>DNA Extraction

The present inventors have extracted DNA from blood of 3 sisters andtheir biological mother with the same method as the above Example <1-1>.

<2-2>Multiplexing Amplification of X-STR

X-STR was amplified by the same method as the above Example <1-2>.

<2-3>Physical and Chemical Characteristics of Amplified STR

Physical and chemical characteristics of amplified DNA were analyzed bythe same method as the above Example <1-3>, and the results were shownin Table 7. TABLE 7 X-STR profiles of mother and daughters Gene ProfileSTR Daughter 1 Daughter 2 Daughter 3 Mother DXS7133 4 5 4 5 4 5 4 6DXS9895 1 1 1 3 1 3 1 1 DXS9898 6 7 6 7 6 7 6 6 DXS6807 2 5 2 5 2 5 2 5DXS6803 2 2 2 3 2 3 2 3 DXS8378 3 4 2 4 2 4 2 3 DXS7132 3 4 4 4 4 4 4 4DXS6789 3 7 7 7 7 7 7 7 GATA31E08 4 5 4 5 4 5 4 4 DXS9900 4 5 4 5 4 5 14 GATA144D04 3 5 3 6 3 6 5 6 GATA165B12 1 3 1 2 1 2 1 2 GATA186D06 3 4 24 2 4 2 3 ATA164A09 5 6 6 6 6 6 1 6 DXS6806 2 3 2 2 2 2 2 3 ATA28C05 3 43 4 3 4 4 5 DXS6804 4 6 4 6 4 6 4 4 DXS6797 2 2 2 3 2 3 2 3<2-4>Comparison of STR Types

Table 7 is showing 18 X-STR gene profiles of three sisters and mother.When mother's genotypes are excluded from sisters', the allelesindicated in dark letters in Table 7 are left. When the two alleles areidentical, both alleles are indicated in same dark letters. In order toshare the same biological father, the alleles indicated in dark letters,in other words alleles of the paternal line, should be identical. Asseen in Table 7, alleles of the paternal line on 18 STR of daughter 2and daughter 3 were all identical, meaning they shared the samebiological parents. It was also confirmed that daughter 1 and daughter 2or daughter 1 and daughter 3 had different biological fathers when theywere sharing the same biological mother.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the present invention provides a novel methodto confirm a blood-relationship that was difficult to be determined withconventional genetic identification. Precisely, the method of thepresent invention can be effectively used to confirm ablood-relationship between suspected-father and suspected daughter bycomparing X-STR types of grandmother and granddaughter or to confirmwhether the suspected-sisters have a same biological father in theabsence of father's DNA. With the method of the present invention, ablood-relationship can be determined easily and fast using the DNAtyping kit of the present invention even in the absence of father's DNA.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A method for determining blood-relationship by typing STR alleles onX chromosome of grandmother and granddaughter, or sisters in the absenceof father's DNA comprising the following steps: 1) extracting DNA fromspecimens of examinee; 2) obtaining massive STR DNA through PCRperformed with STR on X chromosome using the DNA obtained in the abovestep 1 as a template; 3) investigating the physical and chemicalcharacteristics of STR DNA obtained in the above step 2; and 4)determining blood-relationship by comparing types of STR obtained in theabove step
 3. 2. The method for determining blood-relationship as setforth in claim 1, wherein one or more specimens of step 1 can beselected from a group consisting of blood, hair, saliva, epidermis,sperm, samples extracted from vagina, separated cells, tissue samples,dandruffs, ashes, etc, mixed, and used.
 3. The method for determiningblood-relationship as set forth in claim 1, wherein the STR of step 2can be selected partly or altogether from a group consisting of DXS7133,DXS9895, DXS9898, DXS6807, GATA165B12, DXS6797, DXS6803, DXS6806,DXS8378, GATA144D04, DXS7132, DXS6789, GATA186D06, GATA31E08, ATA28C05,GATA172D05, DXS6804, DXS9900 and GATA164A09.
 4. The method fordetermining blood-relationship as set forth in claim 3, wherein the eachSTR is amplified by using primer sets represented by SEQ. ID. No 1-36.5. The method for determining blood-relationship as set forth in claim1, wherein the PCR of step 2 is multiplexing PCR that enablessimultaneous amplification of all different sized 3-10 STR alleles as abundle.
 6. The method as set forth in claim 5, wherein the multiplexingPCR is performed with any panel selected from a group consisting of thefirst panel (DXS7133, DXS9895, DXS9898, DXS6807, GATA165B12 andDXS6797), the second panel (DXS6803, DXS6806, DXS8378, GATA144D04,DXS7132, DXS6789, GATA186D06, GATA31E08 and ATA28C05) and the thirdpanel (GATA172D05, DXS6804 and GATA164A09).
 7. The method fordetermining blood-relationship as set forth in claim 6, wherein theDXS7133, DXS9895, DXS 9898 and DXS6807 of the first panel, DXS6803,DXS6806, DXS8378, GATA144D04 and DXS7132 of the second panel,GATA172D05, DXS6804 and GATA164A09 of the third panel are marked with acertain fluorescent material and GATA165B12 and DXS6797 of the firstpanel, DXS6789, GATA186D06, GATA31E08 and ATA28C05 are marked with theother fluorescent material.
 8. The method for determiningblood-relationship as set forth in claim 1, wherein one of the primersused for the PCR amplification of step 2 is marked with anythingselected from a group consisting of radioisotope, fluorescent material,digoxigenin and biotin.
 9. The method for determining blood-relationshipas set forth in claim 8, wherein one or more fluorescent materials canbe selected from a group consisting of 5-carboxyfluorescein (5-FAM),6-FAM, tetrachlorinated analogue of 6-FAM (TET), hexachlorinatedanalogue of 6-FAM (HEX), 6-carboxytetramethylrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE), NED (ABI), Texas Red™-X, Oregon Green™488 carboxylic acid (Molecular Probes Inc.), Cy-3, Cy-5, Cy-5.5(Amercham PLC), fluorescein-6-isothiocyanate (FITC) andtetramethylrhodamine-5-isothiocyanate (TRITC), and used.
 10. The methodfor determining blood-relationship as set forth in claim 1, wherein thephysical and chemical characteristics of STR are analyzed by the methodselected from a group consisting of the procedure of electrophoresisthat measures the length of DNA fragments amplified by PCR, method thatuse mass spectrometer to measure the mass of the amplified fragments,method that determines the base sequence directly and method that usesDNA array.
 11. The method for determining blood-relationship as setforth in claim 1, wherein the physical and chemical characteristics ofDNA are analyzed by using allele steps of X-STR as a standard.
 12. Themethod for determining blood-relationship as set forth in claim 1,wherein the sisters are sisters by a different mother.
 13. The methodfor determining blood-relationship as set forth in claim 10, wherein theDNA array is provided as the form of DNA sequence of X-STR allele stepsfixed on the surface of supporting material.
 14. A DNA typing kit fordetermining blood-relationship comprising primer sets which are able toamplify the above X-STR, a molecular weight marker consisting of STRallele steps, a general molecular weight marker without STR alleles,amplification enzyme, nucleotides and buffer solution.